PROJECT SUMMARY The main goal of this research proposal is to identify novel ?druggable pathways? to combat castration-resistant prostate cancer (CRPC), the therapy-resistant, lethal form of prostate cancer. There is currently no effective therapy or form of life-extending treatment for this type of prostate cancer. The principal approach of this project is to rigorously scrutinize novel biochemical pathways our lab has implicated in CRPC using a genetic loss-of- function siRNA screen, which enabled the upfront discovery of alterations that cause therapy (i.e. castration) resistance. In addition to known tumor suppressive pathways ? SRC inhibitory kinase CSK and GSK3?, which were further validated in tissue culture studies, animal models, and human prostate cancer tissue samples, the screen implicated several additional pathways. In order to further confirm these results as well as identify additional potential targets, the CRISPR/Cas9 genome-wide knockout system will be utilized with in vivo screening. This method takes advantage of the robust accuracy of the CRISPR/Cas9 system while expanding the range of our screen from only ~700 kinases (as in our previous siRNA screen) to genome-wide. The objective of this study will be addressed in three specific aims: (i.) Specific genes implicated in the progression to CRPC will be identified from a genome-wide loss-of-function (a.) in vivo and (b.) in vitro CRISPR-Cas9-mediated screen (ii.) These hits will be validated (a.) in vitro and (b.) in vivo by assessing the contributions of deficiency of these genes of interest to androgen deprivation-induced growth. The functional role of these pathways will be examined and validated in CRPC mouse models (xenografts, transgenics) as well as in human prostate cancer tissues samples by immunohistochemical staining. Specific cancer biological databases will also be utilized for further confirmation. Not only will these new target pathways be validated functionally, but their activity will also be modified with existing drug-like compounds in order to provide proof- of-concept that they are amenable to pharmacotherapy (i.e. ?druggable?). (iii.) In addition, the underlying causal mechanisms that involve genes of interest and lead to castration resistance will be demystified. Nonconventional activation of the androgen receptor as well as its ectopic expression is a major cause of castration resistance. The mechanisms in which genes of interest accomplish this will be confirmed by combining the use of proteomics and a systems biology approach. Notably, this approach has met with great success in the previous studies on SRC/CSK [9]. The planned investigations are expected to provide a strong rationale for either advancing an existing drug-like compound into clinical development/application or for initiating a screen for small molecules that are able to modulate the pathways implicate in the therapy resistance of CRPC, ultimately addressing a major unmet clinical need in the treatment of CRPC.